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Abstract:

The invention provides for methods of producing pancreatic precursor
cells and insulin-producing cells from bone marrow. In various
embodiments, bone marrow derived stem cells are differentiated into
pancreatic precursor cells and insulin-producing cells. In various
embodiments, bone marrow derived stem cells are artificially induced to
express VEGF and/or PDX-1. These cells can be used to treat or ameliorate
diabetes or symptoms of diabetes.

Claims:

1. A method, comprising: providing a nestin-positive bone marrow stem
cell (BMSC); culturing the nestin-positive BMSC in a first culture medium
for chromatin remodeling by contacting the cell with 5-azacytidine
(5-AZA) and contacting the cell with trichostatin (TSA); culturing the
cell in a second culture medium for cell induction; culturing the cell in
a third culture medium to differentiate the cell; culturing the cell in a
forth culture medium to mature the cell into a pancreatic precursor cell
or an insulin-producing cell.

2. The method of claim 1, wherein the first culture medium comprises one
or more agents selected from the group consisting of KO-DMEM,
β-mercaptoethanol, L-glutamine, non-essential amino acid, BSA, N2
supplement, B27 supplement, heparin, bFGF, EGF, and combinations thereof.

3. The method of claim 1, wherein the second medium comprises one or more
agents from the first culture medium and an agent selected from the group
consisting of DMEM, low glucose, ITS, RA and combinations thereof.

4. The method of claim 1, wherein the third medium comprises one or more
agents from the first culture medium and an agent selected from the group
consisting of DMEM, high glucose and combinations thereof.

5. The method of claim 1, wherein the fourth medium comprises one or more
agents from the first culture medium and an agent selected from the group
consisting of DMEM, high glucose, nicotinamide, and combinations thereof.

6. The method of claim 1, wherein the fourth culture medium does not
contain bFGF or EGF.

7. The method of claim 1, wherein the insulin-producing cell is an
insulin producing β-cell or a β-islet cell.

8. A method, comprising: providing a bone marrow stem cell (BMSC); and
transfecting the BMSC with one or more virus vectors encoding PDX1, VEGF,
or both to produce a VEGF and/or PDX1 expressing BMSC.

11. The method of claim 10, wherein the nucleated cell from the bone
marrow aspirate is cultured in a medium comprising a component selected
from the group consisting of Afla-Men, fetal bovine serum, glutamine,
penicillin, streptomycin and combinations thereof.

12. A method, comprising: providing a bone marrow stem cell (BMSC)
expressing VEGF, a BMSC expressing PDX1 or a BMSC expressing both VEGF
and PDX1; and administering the BMSC to a subject in need of treatment
for diabetes or ameliorating a symptom of diabetes to treat diabetes or
ameliorate the symptom of diabetes.

13. The method of claim 12, wherein: the BMSC expressing VEGF, the BMSC
expressing PDX1 or the BMSC expressing both VEGF and PDX1 is produced by:
isolating a BMSC from a bone marrow sample from the subject; and
artificially increasing the expression of VEGF and/or PDX1 in the BMSC.

14. The method of claim 12, wherein administering is via intravenous
injection of BMSC expressing of VEGF and/or PDX1 into the circulation of
the subject.

15. A method, comprising: providing a pancreatic precursor cell or an
insulin-producing cell differentiated from a bone marrow stem cell
(BMSC); and administering the pancreatic precursor cell or the
insulin-producing cell differentiated from a BMSC to a subject in need of
treatment for diabetes or ameliorating a symptom of diabetes to treat
diabetes or ameliorate the symptom of diabetes.

16. The method of claim 15, further comprising: obtaining bone marrow
from the subject; and producing the pancreatic precursor cell or the
insulin-producing cell from the bone marrow.

18. The method of claim 15, wherein the insulin-producing cell is an
insulin-producing β-cell or a β-islet cell.

19. The method of claim 16, wherein producing the pancreatic precursor
cell or an insulin-producing cell from the bone marrow is by: producing a
nestin-positive bone marrow stem cell (BMSC) from the bone marrow;
culturing the nestin-positive BMSC in a first culture medium for
chromatin remodeling by contacting the cell with 5-azacytidine (5-AZA)
and contacting the cell with trichostatin (TSA); culturing the cell in a
second culture medium for cell induction; culturing the cell in a third
culture medium to differentiate the cell; culturing the cell in a forth
culture medium to mature the cells into a pancreatic precursor cell or an
insulin-producing beta-cell.

20. The method of claim 19, wherein the first culture medium comprises
one or more agents selected from the group consisting of KO-DMEM,
β-mercaptoethanol, L-glutamine, non-essential amino acid, BSA, N2
supplement, B27 supplement, heparin, bFGF, EGF, and combinations thereof.

21. The method of claim 19, wherein the second medium comprises one or
more agents from the first culture medium and an agent selected from the
group consisting of DMEM, low glucose, ITS, RA and combinations thereof.

22. The method of claim 19, wherein the third medium comprises one or
more agents from the first culture medium and an agent selected from the
group consisting of DMEM, high glucose and combinations thereof.

23. The method of claim 19, wherein the fourth medium comprises one or
more agents from the first culture medium and an agent selected from the
group consisting of DMEM, high glucose, nicotinamide, and combinations
thereof.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119(e) to
U.S. Provisional Application No. 61/514,612 filed on Aug. 3, 2011, the
entirety of which is incorporated by reference.

FIELD OF INVENTION

[0002] This invention relates to cell based therapeutic treatments for
diabetes.

BACKGROUND

[0003] All publications herein are incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by reference.
The following description includes information that may be useful in
understanding the present invention. It is not an admission that any of
the information provided herein is prior art or relevant to the presently
claimed invention, or that any publication specifically or implicitly
referenced is prior art.

[0004] Understanding the mechanisms of β-cell homeostasis and
regenerative repair is crucial not only to provide new insights on
diabetes mellitus pathogenesis, but also to address potential therapeutic
application. Islet cell transplantation may be a promising approach for
cell replacement in type 1 diabetes. However, islet availability for
allogeneic transplantation is limited (Lakey et al. 2006, Shapiro et al.
2006). In addition, understanding the origin of new β-cells in
adults may help devise ways of slowing down progression of type 2
diabetes.

[0005] The mechanisms by which β-cell mass is maintained in adulthood
are not yet known and the origin of new β-cells in adults is the
focus of intensive research. The literature is replete with studies
trying to characterize a putative pancreatic progenitor cell in and
outside the pancreas that could explain β-cell neogenesis in adult
life. Should such a cell be identified, it could be used as a potential
source of cell therapy. It has been demonstrated that replacement of
β-cells in the post-natal period precedes both the proliferation of
existing mature β-cells (Dor et al. 2004), and the neogenesis of new
β-cells from progenitor epithelial cells. It has been proposed that
these adult pancreatic progenitor cells reside in the epithelium of the
pancreatic duct (Bouwens & Kloppel 1996, Bonner-Weir 2000, 2001), inside
islets (Zulewski et al. 2001) and in the bone marrow (Janus et al. 2003).

[0006] Nestin is an intermediate filament protein known as a marker of
neuroepithelial stem cells, because it is expressed transiently in early
developmental stages as well as in the process of tissue regeneration in
various organs (Lendahl et al. 1990, Morshead et al. 1994, Matsuoka et
al. 2002). Immunohistochemical studies showed the presence of nestin
positive cells within the islet, the acinar and the ductal compartment of
the prenatal and newborn pancreas (Hunziker & Stein 2000, Zulewski et al.
2001, Kim et al. 2004, Yashpal et al. 2004). The developmental
progression and the change in nestin immunoreactivity throughout the
fetal pancreas to postnatal transition suggest that nestinpositive cells
are probably a population of progenitor cells within the pancreas
(Yashpal et al. 2004). Moreover, Zulewski et al. (2001) described the
existence of a distinct population of cells within islets and in a focal
region of pancreatic ducts and exocrine pancreas expressing nestin that
can proliferate and differentiate into pancreatic, exocrine, ductal and
endocrine cells in culture. A recent publication showed that suppression
of nestin expression in embryonic stem cells by gene silencing reduced
endodermal and pancreatic transcription factor expression (Kim et al.
2010). During embryonic development, neural and islet cells express a
subset of markers in common. Developing islet cells express several
neuronalspecific markers, such as synaptophysins, nerve-specific enolasis
(Alpert et al. 1988), and transcription factor genes such as Isl-1, Pax6,
Pax4, b2/NeuroD, and IDX1 (Reynolds & Weiss 1996, Madsen et al. 1997,
Sander & German 1997).

[0007] Therefore, these data suggest that nestin could be a common marker
of a precursor stem cell for both neuronal and islet cell types. The
inventors' group previously demonstrated that a subpopulation of
nestin-expressing cells, isolated from bone marrow is able to generate
cellular spheres similar to neurospheres derived from brain neural stem
cells. These cells from bone marrow could differentiate into all three
neural phenotypes (neurons, astrocytes, and oligodendrocytes) in vitro
and in vivo (Kabos et al. 2002, Zeng et al. 2007). Recent studies showed
that bone marrow-derived stem cells could reverse the hyperglycemic
phenotype in a diabetic animal model but the mechanism behind the rescue
or regeneration of pancreatic islets is still debated (Hasegawa et al.
2007, Xu et al. 2007, Gao et al. 2008, Zhao et al. 2008).

[0008] Diseases like diabetes are largely caused by a breakdown in cell
function or by cell death. The major issue of diabetes is an inability to
control the level of glucose (sugar) in the blood. Insulin therapy has
saved the lives of many type 1 and type 2 diabetes mellitus patients.
However, 50% of diabetics develop chronic diabetes-related complications
that appear years after the onset of diabetes (including blindness, renal
failure, myocardial infarction, and non-traumatic amputation).

[0009] Despite studies showing that strict blood glucose control decreases
the incidence of secondary complications of diabetes, euglycemia is
difficult to achieve with any current method of exogenous insulin
replacement. Although transplantation of the whole pancreas or islets of
Langerhans demonstrates the physiologic advantages of transplanting
insulin-producing cells over insulin administration, these approaches are
far from perfect. Ideally there are two possible solutions: identifying
the perfect "surrogate β-cell" to be used for cell therapy or
inducing regeneration of endogenous damaged β-cells.

[0010] Stem cells from bone marrow offer an attractive source of stem
cells as alternative to pancreas and pancreatic islets transplantation
for curative and definitive treatment of insulin dependent diabetes. They
are already proved to be safe in clinical trial and they can be obtained
with relative ease from each patient, allowing potential circumvention of
allograft rejection. Previous work suggested that the mouse BMSCs
spontaneously differentiate into endocrine pancreas cells in vivo. In
recent reports, BMSCs injected into the circulation of diabetic animal
has been shown to partially/totally reverse the diabetic phenotype and
improve glucose control, but with very poor direct β-cell
differentiation, leading to other possible roles of BMSCs in the
pancreatic islet regeneration. According, additional treatment methods
are needed in the art.

[0011] Encouragingly, as further described herein the introduction of
transcription factor genes into cultured human BMSCs was able to activate
a number of genes related with development and function of β-cells.
Moreover, the forced expression of Pdx1 gene by a virus vector in human
BMSCs showed the activation of gene expression of all four islets
hormones and also enhancement of significant insulin content.

SUMMARY OF THE INVENTION

[0012] The following embodiments and aspects thereof are described and
illustrated in conjunction with compositions and methods which are meant
to be exemplary and illustrative, not limiting in scope.

[0013] Various embodiments of the present invention provide for a method,
comprising: providing a nestin-positive bone marrow stem cell (BMSC);
culturing the nestin-positive BMSC in a first culture medium for
chromatin remodeling by contacting the cell with 5-azacytidine (5-AZA)
and contacting the cell with trichostatin (TSA); culturing the cell in a
second culture medium for cell induction; culturing the cell in a third
culture medium to differentiate the cell; culturing the cell in a forth
culture medium to mature the cell into a pancreatic precursor cell or an
insulin-producing cell. In various embodiments, the insulin-producing
cell is an insulin producing β-cell or a β-islet cell.

[0014] In various embodiments, the first culture medium can comprise one
or more agents selected from the group consisting of KO-DMEM,
β-mercaptoethanol, L-glutamine, non-essential amino acid, BSA, N2
supplement, B27 supplement, heparin, bFGF, EGF, and combinations thereof.
In various embodiments, the second medium can comprise one or more agents
from the first culture medium and an agent selected from the group
consisting of DMEM, low glucose, ITS, RA and combinations thereof. In
various embodiments, the third medium can comprise one or more agents
from the first culture medium and an agent selected from the group
consisting of DMEM, high glucose and combinations thereof. In various
embodiments, the fourth medium can comprise one or more agents from the
first culture medium and an agent selected from the group consisting of
DMEM, high glucose, nicotinamide, and combinations thereof. In various
embodiments, the fourth culture medium does not contain bFGF or EGF.

[0015] Various embodiments of the present invention provide for a method,
comprising: providing a bone marrow stem cell (BMSC); and transfecting
the BMSC with one or more virus vectors encoding PDX1, VEGF, or both to
produce a VEGF and/or PDX1 expressing BMSC. In various embodiments, the
virus can be an adenovirus.

[0016] In various embodiments, the BMSC can be obtained by: providing bone
marrow aspirate; culturing a nucleated cell from the bone marrow
aspirate; separating a non-adherent cell from an adherent cell; culturing
the adherent cell; harvesting a bone marrow stem cell; and expanding the
bone marrow stem cell. In various embodiments, the nucleated cell from
the bone marrow aspirate may be cultured in a medium comprising a
component selected from the group consisting of Afla-Men, fetal bovine
serum, glutamine, penicillin, streptomycin and combinations thereof.

[0017] Various embodiments of the present invention provide for a method,
comprising: providing a bone marrow stem cell (BMSC) expressing VEGF, a
BMSC expressing PDX1 or a BMSC expressing both VEGF and PDX1; and
administering the BMSC to a subject in need of treatment for diabetes or
ameliorating a symptom of diabetes to treat diabetes or ameliorate the
symptom of diabetes. In various embodiments, administering can be via
intravenous injection of BMSC expressing of VEGF and/or PDX1 into the
circulation of the subject.

[0018] In various embodiments, the BMSC expressing VEGF, the BMSC
expressing PDX1 or the BMSC expressing both VEGF and PDX1 can be produced
by: isolating a BMSC from a bone marrow sample from the subject; and
artificially increasing the expression of VEGF and/or PDX1 in the BMSC.

[0019] Various embodiments of the present invention provide for a method,
comprising: providing a pancreatic precursor cell or an insulin-producing
cell differentiated from a bone marrow stem cell (BMSC); and
administering the pancreatic precursor cell or the insulin-producing cell
differentiated from a BMSC to a subject in need of treatment for diabetes
or ameliorating a symptom of diabetes to treat diabetes or ameliorate the
symptom of diabetes. In various embodiments, administering can be via
intra-hepatic or subcutaneous transplantation.

[0020] In various embodiments, the method can further comprise obtaining
bone marrow from the subject; and producing the pancreatic precursor cell
or the insulin-producing cell from the bone marrow. In various
embodiments, the insulin-producing cell can be an insulin-producing
β-cell or a β-islet cell.

[0021] In various embodiments, producing the pancreatic precursor cell or
an insulin-producing cell from the bone marrow can be by: producing a
nestin-positive bone marrow stem cell (BMSC) from the bone marrow;
culturing the nestin-positive BMSC in a first culture medium for
chromatin remodeling by contacting the cell with 5-azacytidine (5-AZA)
and contacting the cell with trichostatin (TSA); culturing the cell in a
second culture medium for cell induction; culturing the cell in a third
culture medium to differentiate the cell; culturing the cell in a forth
culture medium to mature the cells into a pancreatic precursor cell or an
insulin-producing beta-cell.

[0022] In various embodiments, the first culture medium can comprise one
or more agents selected from the group consisting of KO-DMEM,
β-mercaptoethanol, L-glutamine, non-essential amino acid, BSA, N2
supplement, B27 supplement, heparin, bFGF, EGF, and combinations thereof.
In various embodiments, the second medium can comprise one or more agents
from the first culture medium and an agent selected from the group
consisting of DMEM, low glucose, ITS, RA and combinations thereof. In
various embodiments, the third medium can comprise one or more agents
from the first culture medium and an agent selected from the group
consisting of DMEM, high glucose and combinations thereof. In various
embodiments, the fourth medium can comprise one or more agents from the
first culture medium and an agent selected from the group consisting of
DMEM, high glucose, nicotinamide, and combinations thereof.

[0023] Other features and advantages of the invention will become apparent
from the following detailed description, taken in conjunction with the
accompanying drawings, which illustrate, by way of example, various
features of embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

[0024] Exemplary embodiments are illustrated in referenced figures. It is
intended that the embodiments and figures disclosed herein are to be
considered illustrative rather than restrictive.

[0026] FIG. 2 depicts engraftment of hBMSC-VEGF in the injured pancreas in
accordance with various embodiments of the present invention.
Fluorescence immunostaining for human β-2-microglobulin (green)
shows engraftment of the hBMSC-VEGF in the pancreas of rescued mice (A).
At higher magnification it is evident the differentiation to vascular and
ductal structures in the pancreas (B). Fluorescence immunestaining for
β-2-microglobulin shows more modest human stem cell engraftment in
the pancreas of the unrescued mice (C). Panel 2D shows few cells in a
pancreatic islet co-staining with human β-2-microglobulin (green)
and insulin (red), and small vessel like structure inside the pancreas
co-staining with β-2-microglobulin (green) and a smooth muscle actin
(red). Scale bar: 50 μm. Panel E shows the percent of the pancreatic
islets expressing human β2-microglobulin, and the percent of
β-cells expressing human β2-microglobulin in the positive
pancreatic islets (Right panel). Additionally, fluorescence
immunostaining for human β-2-microglobulin (green) showed
engraftment of the hBMSC-VEGF in the mesenchymal tissue outside the
pancreas (F). At higher magnification it is evident the differentiation
in vascular and ductal structure in vascular and adipose tissue in the
mesenchymal outside the pancreas (G).

[0027] FIG. 3 depicts VEGF expression in the pancreatic islets in
accordance with various embodiments of the present invention. Fluorescent
staining for VEGF (red) and insulin (green) in the pancreatic islets of
control mice (A-C), diabetic mice (STZ;D-F), and diabetic mice rescued by
hBMSC-VEGF (G-I). Arrows (F) show only few cells co-expressing insulin
and VEGF. Percentage of β-cells expressing VEGF in the pancreas of
control mice, diabetic mice (STZ) and mice rescued by hBMSC-VEGF (J).
Scale bar: 50 μm. **p<0.001.

[0030] FIG. 6 depicts serum insulin and β-cell number in accordance
with various embodiments of the present invention. Both healthy control
mice and diabetic mice rescued by hBMSC-VEGF (hBM-VEGF-R) have
significantly higher mouse insulin level (A) compared with diabetic mice
(STZ), diabetic mice treated with hBMSC (hBM), diabetic mice unrescued by
hBMSC-VEGF (hBM-VEGF-Ur), and diabetic mice treated with hBMSC-PDX1
(hBM-PDX1). There is no significant difference between mouse insulin
level of healthy control mice and diabetic mice rescued by hBMSC-VEGF
(hBM-VEGF-R). Human insulin is detected in 3 rescued mice treated with
hBMSC-VEGF, 2 unrescued mice treated with hBMSC-VEGF, and 5 mice treated
with hBMSC-PDX1 (B). Diabetic mice rescued by hBMSC-VEGF (hBM-VEGF-R)
have significantly higher total insulin level (human and mouse insulin)
(C) compared with diabetic mice (STZ), diabetic mice treated with hBMSC
(hBM), diabetic mice unrescued by hBMSC-VEGF (hBM-VEGF-Ur), and diabetic
mice treated with hBMSC-PDX1 (hBM-PDX1), and not significantly different
from control healthy mice. Mice unrescued by hBMSC-VEGF and mice treated
with hBMSC-PDX1 have significant higher total insulin level compared with
diabetic mice and diabetic mice treated with hBMSC, but lower than
control mice and mice rescued by hBMSC-VEGF. β-cell number is higher
in the healthy control mice compared with other groups (D). Mice rescued
by hBMSC-VEGF have significant higher β-cell number than diabetic
mice, diabetic mice treated with hBMSC, diabetic mice unrescued by
hBMSC-VEGF, and diabetic mice treated with hBMSC-PDX1, but significantly
lower than healthy control mice. Diabetic mice unrescued by hBMSC-VEGF
and treated with hBMSC-PDX1 have significantly higher β-cell number
than diabetic mice and diabetic mice treated with hBMSC, but
significantly lower than mice rescued by hBMSC-VEGF. *p<0.05,
**p<0.01, ***p<0.001.

[0034] FIG. 10 depicts expression of nestin and pancreatic transcription
factors during in vitro n-BMSC differentiation in accordance with various
embodiments of the present invention. (A-D) Immunofluorescence staining
for nestin was positive (A) in the beginning and negative in the
following differentiation step. (E-H) Immunofluorescence for PDX1 was
negative in the beginning (A) and positive in the three following steps.
(I-L) Immunofluorescence staining for PAX6 was negative in the beginning
(A); positive in only few cells by step 2 (J), and in many cells by step
3 (K); negative again in the last step (L). Nuclei were counterstained
with DAPI. Confocal microscopy, original magnification ×63 (A, F,
G, and H); Fluorescent microscopy ×40 (K) and ×20 (J and L).

[0035] FIG. 11 depicts quantitative reverse transcription-PCR analysis of
n-BMSC for Pdx1 and Ngn3 in accordance with various embodiments of the
present invention. (A): Pdx1 and Ngn3 genes were not expressed in the
beginning of pancreatic differentiation in vitro but were induced during
step 2 of the differentiation protocol. DCT presents the difference of
the cycle threshold between the assayed gene and the normalizing gene.
(B) Variation of gene expression during in vitro differentiation. Values
are fold change GS.D. (nZ3). *P<0.05; **P<0.01.

[0036] FIG. 12 depicts differentiation of n-BMSC in a mature pancreatic
phenotype in accordance with various embodiments of the present
invention. Immunofluorescence staining for CK19 (A and B) and for insulin
(C and D) was negative until the third differentiation step and became
positive in the last step (B and D). Dithizone staining for proinsulin (E
and F) was also positive in the last differentiation step. Nuclei were
counterstained with DAPI. Images of insulin staining visualized under
confocal microscope (D-G) with detail of single-cell sectioning (H)
showing the insulin granular cytoplasmatic pattern. Confocal microscopy,
original magnification ×63 (B, D, G, and H); phase contrast
microscopy, original magnification ×20 (E and F).

[0037] FIG. 13 depicts quantitative reverse transcription-PCR analysis of
n-BMSC in accordance with various embodiments of the present invention.
(A) Gene not expressed in the beginning of pancreatic differentiation in
vitro and induced during step 3 of the differentiation protocol. DCT
represents the difference between the cycle threshold of the assayed gene
and that of the normalizing gene. (B) Variation of gene expression during
in vitro differentiation. (C) Insulin production in response to glucose
concentration: n-BMSCs were incubated in a buffer containing the
indicating concentration of glucose. Values are fold change±S.D.
(n=3). **P<0.01; ***P<0.005; ****P<0.0001.

[0038] FIG. 14 depicts blood glucose level after hBMSC transplantation in
accordance with various embodiments of the present invention. (A):
healthy control mice (rectangle), STZ-induced diabetes mice (black
square), and STZ-induced diabetes mice after treatment with hBMSC (white
triangle). Pancreases immunohistochemistry (B): H&E staining showed
normal pancreatic islets number, size and morphology in the healthy
control mice, compared with reduced size and altered morphology of the
diabetic mice with (STZ+hBMSC) or without hBMSC treatment (STZ).
Fluorescence staining for insulin (green) and glucagon (red) of the
control pancreas, compared with the diabetic mice with (STZ+hBMSC) or
without hBMSC (STZ). Scale bare: 50 μm.

DESCRIPTION OF THE INVENTION

[0039] All references cited herein are incorporated by reference in their
entirety as though fully set forth. Unless defined otherwise, technical
and scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this invention
belongs. Singleton et al., Dictionary of Microbiology and Molecular
Biology 3rd ed., J. Wiley & Sons (New York, N.Y. 2001); March,
Advanced Organic Chemistry Reactions, Mechanisms and Structure 5th
ed., J. Wiley & Sons (New York, N.Y. 2001); and Sambrook and Russel,
Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor
Laboratory Press (Cold Spring Harbor, N.Y. 2001), provide one skilled in
the art with a general guide to many of the terms used in the present
application.

[0040] One skilled in the art will recognize many methods and materials
similar or equivalent to those described herein, which could be used in
the practice of the present invention. Indeed, the present invention is
in no way limited to the methods and materials described. For purposes of
the present invention, the following terms are defined below.

[0041] "Mammal" as used herein refers to any member of the class Mammalia,
including, without limitation, humans and nonhuman primates such as
chimpanzees, and other apes and monkey species; farm animals such as
cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and
cats; laboratory animals including rodents such as mice, rats and guinea
pigs, and the like. The term does not denote a particular age or sex.
Thus adult and newborn subjects, as well as fetuses, whether male or
female, are intended to be including within the scope of this term.

[0042] "Treatment" and "treating," as used herein refer to both
therapeutic treatment and prophylactic or preventative measures, wherein
the object is to prevent, slow down and/or lessen the disease even if the
treatment is ultimately unsuccessful.

[0043] Described herein is a novel culture system in order to obtain
insulin producing cells derived from nestin-positive bone marrow cells
(n-BMSC). This procedure successfully induced n-BMSC to express
lineage-specific transcription factor, in particular Pdx1, which has been
shown to drive pancreatic differentiation.

[0044] The cells were pretreated with 5-AZA to inhibit DNA methylation,
which has been recognized in important differentiation processes, such as
osteoblast (Vaes et al. 2009), cardiomycyte (Tomita et al. 1999) and more
recently pancreatic endocrine differentiation (Lefebvre et al. 2009). The
cells were subsequently treated with TSA based on the belief that
chromatin remodeling will enhance the induction and further
differentiation toward pancreatic lineage, although not wishing to be
bound by any particular theory. Acetylation or deacetylation of histone
terminal domains can regulate gene expressions. Histone
acetyltransferases and HDACs can loosen or compact chromatin structures
and regulate cell proliferation and differentiation in various tissues
(Zhang et al. 2002, Chang et al. 2004). As the acetylation status of
nucleosomal histone modulates chromatin structure and epigenetically
regulates gene expression, an inhibitor of both classes I and II HDACs
such as TSA may be used to loosen the chromatin structure and more easily
permit differentiation through epigenetic manipulation. Moreover, a
recent study from Haumaitre et al. (2008) showed that HDAC inhibitors can
modify the timing and the determination of pancreatic cell fate with
inhibition of exocrine and enhancement of ductal and endocrine
differentiation. In the experiment described herein, after exposure to
5-AZA and TSA, the cells lost nestin expression and after being cultured
in a medium containing ITS and RA, they also turned on expression of mRNA
encoding transcription factors such as Pdx1, NgN3 and Pax6, known to play
important roles in the developmental program leading to the formation of
pancreatic islets (Jensen 2004). Moreover, the expression of these
transcription factors followed the timing consistent with the normal
mammalian pancreatic development. PDX1 was expressed in the second step
after exposure to RA and ITS medium and subsequently expression of both
PAX6 and NGN3 were activated. In the last step, a more mature phenotype
of ductal and endocrine pancreatic cells appeared. These cells lost the
expression of PAX6, and upregulated PDX1, insulin, glucagon, GLUT2 and
CK19.

[0045] Another important step in this differentiation protocol is the use
of RA to induce differentiation of n-BMSC towards a pancreatic phenotype.
In the experiment described herein, it was extremely important to time
the exposure to RA, right after the cell treatment with TSA. RA has been
shown to control multiple steps in the motor neuron differentiation in
the ventral spinal cord (Novitch et al. 2003). In an experiment, Ostrom
et al. (2008) showed that RA is present in the developing mouse and human
pancreas and is required for pancreas development. Moreover, all-trans RA
has been widely used to promote generation of PDX1-positive pancreatic
progenitor cells from mouse embryonic stem cells (Micallef et al. 2005,
Shi et al. 2005) and to induce pancreatic differentiation in human
embryonic stem cells (D'Amour et al. 2006, Johannesson et al. 2009). In
the experiment described herein, consistent with the previous
observations, it is shown showed that RA induces expression of PDX1.

[0046] In the last step both ductal and endocrine phenotypes were present
and this is probably because the activation of Pdx1 in the second step of
the protocol, subsequently activated both the endocrine and the
non-endocrine pathways. Pdx1 is well known for controlling early whole
pancreas development.

[0047] The inventors believe, although not wishing to be bound by any
particular theory, that a selected stem cell population from rat whole
bone marrow highly expressing nestin could be a potential multipotent
precursor stem cell able to differentiate to pancreatic cell lineage.
This belief was based on the evidence that nestin is a neuroepithelial
marker transiently expressed in early stages in many tissues, including
pancreas, and that nestin-positive cells from pancreas can be
differentiated in culture into endocrine, exocrine, and ductal cells
(Zulewski et al. 2001).

[0048] The inventors demonstrated that epigenetic manipulation of n-BMSC,
following a specific multistep protocol, can induce expression of
transcription factors involved in the early pancreatic and endocrine
specification in a step-wise fashion whose timing was consistent with
normal pancreas development. Differentiation into a more mature
phenotype, including ductal and insulin-producing cells was efficiently
induced. It remains to be shown how long these cells remain in this
differentiated state both in vitro and in vivo. In conclusion, the
inventors describe a novel cellular system that can address these unmet
needs, both for cell therapy and for mechanistic studies and drug
discovery in the field of pancreatic islet neogenesis.

[0049] Moreover, the inventors were able to induce reversion of diabetes
for genetic modification of bone marrow stem cells using PDX1 and VEGF
gene. This is the first time that stem cell from bone marrow expressing
VEGF has been used as possible treatment of type 1 Diabetes Mellitus.
After transplantation in a diabetic animal model, the inventors observed
de novo differentiation of human bone marrow stem cells into β-cells
and for the first time in the literature the inventors report detectable
level of human insulin, confirming a successful chimerism and a
functional differentiation of human bone marrow stem cells in beta-cells.

[0050] Stem cell therapy may be a desirable alternative to pancreas and
pancreatic islet transplantation, and stem cells from bone marrow
represent an attractive source. However, the mechanism related to
β-cell recovery required elucidation. Here, for the first time the
inventors tested the hypothesis of de novo β-cell differentiation
from hBMSCs versus endogenous β-cell regeneration mediated by
hBMSCs, using the transient expression of PDX1 and VEGF.

[0051] In contrast to previous reports [3,7,8,9,10,11] hBMSCs alone were
not able to reverse hyperglycemia in the inventors' animal model. This
can be attributed to differences in stem cell populations, mouse strains,
mouse models, and experimental designs among research groups. Some
studies used hematopoietic stem cells from bone marrow [3,32], or mouse
mesenchymal bone marrow stem cells [8,9,10,11]. In addition C57BL/6 were
used for STZ-induced diabetes model [8,10,11], which can explain
different outcomes in response to STZ, degree of diabetes, blood glucose
levels and responses to treatment. Only one previous report [7] is the
closest to the inventors' study: same type of stem cells, same cell
delivery method (intracardiac injection) and same mouse strain. However
the dose of STZ used was adjusted to produce nonlethal hyperglycemia and
an improvement in blood glucose control was possible only with multiple
injections of human cells. The higher glucose level and mortality of the
inventors' mice in addition to a single stem cell injection can explain
the different results.

[0052] The inventors achieved sustained recovery from diabetes following
injection of hBMSCs overexpressing VEGF. The inventors observed an
efficient engraftment of hBMSC-VEGF in the pancreas of the diabetic mice,
and its successful differentiation into blood vessels and to lesser
degree into β-cells. For the first time, the inventors reported
detectable levels of human insulin confirming a successful chimerism in
the mouse. However, the sustained near-normoglycemia remission was not
fully supported by the low level of human insulin, and the low number of
β-cells from hBMSCs-VEGF. The significantly higher level of mouse
insulin in rescued mice suggested that the endogenous β-cell
regeneration was the predominant mechanism behind the sustained clinical
recovery. Taken together the de novo intra pancreatic angiogenesis from
hBMSC-VEGF along with the endogenous activation of the insulin/IGF
receptor signaling pathway strongly support regeneration and functional
recovery of endogenous β-cells.

[0053] To highlight this concept, the inventors performed a parallel
experiment using hBMSCs expressing PDX1. The previous report showed the
possible direct differentiation of human hBMSCs into β-cells in
vitro after transfection with a virus vector encoding PDX1[17],
supporting the possible role of PDX1 to direct the differentiation of
hBMSCs into insulin-producing cells in the inventors' in vivo model.
Human BMSC-PDX1 could differentiate to β-cells in the diabetic
pancreas with approximately the same efficiency of hBMSC-VEGF confirmed
by similar detectable levels of serum human insulin and by
immunohistochemistry. However, transplantation of hBMSC-PDX1 into
diabetic mice resulted in only transient recovery. The overall efficiency
of hBMSC-PDX1 engraftment and their differentiation into blood vessels
were significantly lower than that from hBMSC-VEGF, which is correlated
with the disparate clinical outcomes. VEGF-A has been known to play a key
role in maintaining normal intra-islet vascularization [33,34]. In
addition, VEGF is known to enhance proliferation, survival and
differentiation of bone marrow mesenchymal stem cells [35]. Over
expression of VEGF in BMSC increased revascularization and myocardial
recovery after injury [36], and neutralizing anti-VEGF antibodies
inhibited the BMSC-initiated angiogenic response in vivo [37]. Moreover
the β-cell-specific VEGF-A deficient mouse showed the altered
insulin secretion despite maintaining a normal β-cell mass [38] and
bone marrow transplantation did not induce expansion of β-cell mass
after STZ-induced diabetes compared with wild-type mice [39]. It has been
reported that BMSCs improved revascularization and function of pancreatic
islets after transplantation [40]. Bone marrow mesenchymal stem cells can
not only promote endogenous angiogenesis [41], but directly differentiate
into smooth muscle [42] and endothelial cell phenotypes [43] in vitro,
and into functional vascular structures [44,45] and contribute in vivo to
myocardial recovery after injury [46].

[0054] The pancreas of the mice rescued by hBMSC-VEGF showed upregulation
of insulin receptor associated gene, such as Ins1, Igf2, Igfbp1 as well
as Dok1, 2 and 3. Recent extensive studies have shown the importance of
insulin regulating β-cell function [47]. The inventors' results
showed that Insulin/IGF receptor coupling with insulin receptor soluble
(IRS) proteins activated the downstream effector pathway PI-3K. Several
gene targets in the PI-3K pathway were upregulated including Adra1d, G6
pc, G6 pc2, and Serpine 1 in the rescued group. In contrast, Grb2,
generally thought to affect Ras and mitogen-activated protein kinase
signaling, was significantly downregulated in the rescued mice.
Consistent with a previous report [48], the expression of Jun was
increased in the diabetic pancreas while it was significantly
downregulated in the rescued mice. Insulin/IGF receptor/PI-3K signaling
mediates several pathways related to proliferation and anti-apoptosis in
most mammalian cells including pancreatic islets [49]. AKT is a critical
mediator of the Insulin/IGF receptor/PI-3K pathway and overexpression of
active AKT1 in the mouse β-cells significantly increased β-cell
size and total islet mass [50]. The inventors showed a significant
decrease of AKT expression in the pancreatic islets of diabetic mice,
compared with control and rescued mice. Interestingly, pattern of AKT
distribution was mostly on the cell membrane of the β-cells of the
healthy control mice, while it was highly expressed in both cell membrane
and cytoplasm of the β-cells in the rescued mice. It is well known
that AKT activation takes place on the cell membrane [51]. On the other
hand, it has been reported that translocation of AKT in the cytoplasm and
nucleus after stimulation with growth factors such us insulin and IGF1
could mediate potential anti-apoptotic mechanisms [52,53]. Activation of
Insulin/IGF signaling through PI-3K/ATK pathway could induce reduction of
apoptosis by cytoplasmic sequestration of BAD that prevents BCL2
activation and subsequently caspase activation. Thus a reduced
phosphorylation of BAD in diabetic pancreas is consistent with increased
apoptosis [54]. The inventors' data also showed increased apoptosis in
the diabetic mouse pancreas measured by the increased number of
β-cells expressing caspase 3 cleaved. Accordingly, diabetic mice
rescued by hBMSC-VEGF showed a significant reduction in apoptosis.

[0055] In keeping with the observation of enhanced insulin/PI-3K/AKT
signaling, the pancreatic islets of the rescued mice showed greater
expression and nuclear localization of PDX1 compared with diabetic mice.
PDX1 is a downstream transcriptional target of insulin signaling and it
is required for β-cell growth and differentiation [27]. Moreover the
expression level of p27.sup.Kip1, a cell cycle inhibitor known to be
negatively regulated by PI-3K/AKT pathway in β-cells through
FoxO1[31] and Gsk-3β[30], was significantly reduced in the rescued
group while up-regulated in control and diabetic pancreatic islets. This
finding confirmed the increased proliferative signal in the rescued
pancreatic islets through the Insulin receptor/PI-3K/ATK pathway,
compared with both control healthy mice and diabetic mice.

[0056] In addition, PI-3K signaling is also known to modulate VEGF
expression in the endothelial cells and to induce angiogenesis [29]
though v-Src [55]. Moreover, VEGF expression is predominant in the
pancreatic β-cells and VEGF receptor 2 (VEGFR2) is highly expressed
in the intra-islets capillary [20]. The inventors' data showed a dramatic
increase in VEGF expression in the β-cells of rescued mice compared
with diabetic mice, implying the possible activation of VEGF expression
via PI-3K/AKT pathway.

[0057] Taking together, the inventors' results suggest that hBMSCs-VEGF
induce reversion of diabetes mainly by induction of endogenous
β-cell regeneration through the generation of a favorable
microenvironment and through the activation of the insulin/IGF1/PI-3K/AKT
pathway. Activation of this pathway in the β-cells improves cell
survival through inhibition of apoptosis and induces β-cell
differentiation and proliferation through activation of PDX1 expression
and inhibition of P27.sup.Kip1. In addition, the inventors provide
evidence of a possible new mechanism of β-cell recovery/regeneration
through modulation of intra-islet angiogenesis. The activation of the
insulin/IGF signaling through the PI-3K pathway in the diabetic mice
rescued by hBMSC-VEGF induces VEGF expression in the β-cells and
correlated with β-cell recovery (FIG. 8F).

[0058] In conclusion, the inventors' work provides new insight into the
mechanism of β-cell recovery after injury mediated by hBMSC therapy
and demonstrates the th ability to use hBMSC expressing VEGF for the
treatment of insulin-dependent diabetes.

[0059] Embodiments of the present invention are based, at least in part,
by these findings.

[0060] Various embodiments of the present invention provide for a method
of producing pancreatic precursor cells from whole bone marrow and
insulin-producing cells from whole bone marrow. The method can comprise
chromatin remodeling, induction, differentiation and maturation.

[0061] Chromatin remodeling can comprise providing nestin-positive bone
marrow-derived neurospheres, culturing the nestin-positive bone
marrow-derived neurospheres in a first culture medium (basal medium);
contacting the nestin-positive bone marrow-derived neurospheres with
5-AZA; changing the first culture medium; and contacting the cells with
TSA. In various embodiments, the first culture medium can comprise a
component selected from the group consisting of KO-DMEM,
β-mercaptoethanol, L-glutamine, non-essential amino acid, BSA, N2
supplement, B27 supplement, heparin, bFGF, EGF, and combinations thereof.

[0062] Induction can comprise changing the first medium to a second medium
(induction medium), culturing the cells for about 7 days. In various
embodiments, the second culture medium can comprise one or more
components from the first culture medium and a component selected from
the group consisting of DMEM, low glucose, ITS, RA and combinations
thereof.

[0063] Differentiation can comprise culturing the cells in a third medium
(differentiation medium) for about 7 days. In various embodiments, the
third medium can comprise one or more components from the first culture
medium and a component selected from the group consisting of DMEM, high
glucose and combinations thereof.

[0064] Maturation can comprise culturing the cells in a forth medium
(maturation medium) for about 7 days. In various embodiments, the fourth
medium can comprise one or more components from the first culture medium
and a component selected from the group consisting of DMEM, high glucose,
nicotinamide, and combinations thereof. In various embodiments, the forth
medium does not contain bFGF and EGF.

[0065] In various embodiments, the first, second, third and fourth media
are changed about every 2 days within each step.

[0066] In various embodiments, β-islet cells and particularly,
insulin producing β-cells are produced by these methods.

[0067] Various embodiments of the present invention provide for pancreatic
precursor cells derived from whole bone marrow and insulin-producing
cells from whole bone marrow. In various embodiments the pancreatic
precursor cells are produced by methods of the present invention. Other
embodiments of the present invention provide for β-islet cells and
insulin producing β-cells are produced by methods of the present
invention.

[0068] Various embodiments of the present invention provide for a method
of producing VEGF and PDX1 expressing bone marrow stem cells.

[0069] The method can comprise providing bone marrow stem cells and
transfecting the BMSCs with a virus carrying PDX1, VEGF, or both. In
various embodiments, the virus is an adenovirus.

[0070] In various embodiments, bone marrow stem cells can be obtained by
providing bone marrow aspirate, culturing the nucleated cells from the
bone marrow aspirate in a culture medium; separating the non-adherent
cells from the adherent cells; washing the adherent cells and culturing
the adherent cells; harvesting the cells and expanding the cells. In
various embodiments, the culture medium can comprise a component selected
from the group consisting of Afla-Men, fetal bovine serum, glutamine,
penicillin, streptomycin and combinations thereof.

[0071] Various embodiments of the present invention provide for treatment
of diabetes and ameliorating symptoms of diabetes.

[0072] In various embodiments, the method can comprise, providing
insulin-producing β-cells differentiated in vitro from BMSC, BMSC
expressing VEGF, BMSC expressing PDX1, or BMSC expressing both VEGF and
PDX1, and administering the BMSC, the BMSC expressing VEGF, PDX1 or both
to a subject in need thereof. In various embodiments, the BMSC expressing
VEGF and/or PDX1 are BMSCs that are artificially induced to express VEGF
and/or PDX1; for example, by transfection of an adenovirus vector
expressing VEGF and/or PDX1.

[0073] In various embodiments, the method can comprise obtaining bone
marrow from a subject in need thereof, producing insulin-producing
β-cells from the bone marrow, and transplanting the
insulin-producing β-cells back into the same subject. In various
embodiments, transplantation could be intra-hepatic or subcutaneous. In
various embodiments, producing insulin-producing beta-cells from the bone
marrow may be through the methods of the present invention.

[0074] In various embodiments, the method can comprise isolating BMSC from
a bone marrow sample from a subject; genetically modifying the BMSC to
increase or induce expression of VEGF and/or PDX1, and transplantation of
BMSC into the circulation of the same subject by intravenous injection.
In various embodiments, the BMSCs are undifferentiated. Methods of
genetically modifying the BMSCs to increase or induce expression of a
gene in a cell are known in the art and can readily be used in the
present invention.

[0075] In various embodiments, the present invention provides
pharmaceutical compositions including a pharmaceutically acceptable
excipient along with a therapeutically effective amount of pancreatic
precursor cells or insulin-producing cells derived from whole bone
marrow. "Pharmaceutically acceptable excipient" means an excipient that
is useful in preparing a pharmaceutical composition that is generally
safe, non-toxic, and desirable, and includes excipients that are
acceptable for veterinary use as well as for human pharmaceutical use.
Such excipients may be solid, liquid, semisolid, or, in the case of an
aerosol composition, gaseous.

[0076] In various embodiments, the pharmaceutical compositions according
to the invention may be formulated for delivery via any route of
administration. "Route of administration" may refer to any administration
pathway known in the art, including but not limited to aerosol, nasal,
oral, transmucosal, transdermal or parenteral. "Transdermal"
administration may be accomplished using a topical cream or ointment or
by means of a transdermal patch. "Parenteral" refers to a route of
administration that is generally associated with injection, including
intraorbital, infusion, intraarterial, intracapsular, intracardiac,
intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal,
intrasternal, intrathecal, intrauterine, intravenous, subarachnoid,
subcapsular, subcutaneous, transmucosal, or transtracheal. Via the
parenteral route, the compositions may be in the form of solutions or
suspensions for infusion or for injection, or as lyophilized powders. Via
the enteral route, the pharmaceutical compositions can be in the form of
tablets, gel capsules, sugar-coated tablets, syrups, suspensions,
solutions, powders, granules, emulsions, microspheres or nanospheres or
lipid vesicles or polymer vesicles allowing controlled release. Via the
parenteral route, the compositions may be in the form of solutions or
suspensions for infusion or for injection. Via the topical route, the
pharmaceutical compositions based on compounds according to the invention
may be formulated for treating the skin and mucous membranes and are in
the form of ointments, creams, milks, salves, powders, impregnated pads,
solutions, gels, sprays, lotions or suspensions. They can also be in the
form of microspheres or nanospheres or lipid vesicles or polymer vesicles
or polymer patches and hydrogels allowing controlled release. These
topical-route compositions can be either in anhydrous form or in aqueous
form depending on the clinical indication.

[0077] The pharmaceutical compositions according to the invention can also
contain any pharmaceutically acceptable carrier. "Pharmaceutically
acceptable carrier" as used herein refers to a pharmaceutically
acceptable material, composition, or vehicle that is involved in carrying
or transporting a compound of interest from one tissue, organ, or portion
of the body to another tissue, organ, or portion of the body. For
example, the carrier may be a liquid or solid filler, diluent, excipient,
solvent, or encapsulating material, or a combination thereof. Each
component of the carrier must be "pharmaceutically acceptable" in that it
must be compatible with the other ingredients of the formulation. It must
also be suitable for use in contact with any tissues or organs with which
it may come in contact, meaning that it must not carry a risk of
toxicity, irritation, allergic response, immunogenicity, or any other
complication that excessively outweighs its therapeutic benefits.

[0078] The pharmaceutical compositions according to the invention can also
be encapsulated, tableted or prepared in an emulsion or syrup for oral
administration. Pharmaceutically acceptable solid or liquid carriers may
be added to enhance or stabilize the composition, or to facilitate
preparation of the composition. Liquid carriers include syrup, peanut
oil, olive oil, glycerin, saline, alcohols and water. Solid carriers
include starch, lactose, calcium sulfate, dihydrate, terra alba,
magnesium stearate or stearic acid, talc, pectin, acacia, agar or
gelatin. The carrier may also include a sustained release material such
as glyceryl monostearate or glyceryl distearate, alone or with a wax.

[0079] The pharmaceutical preparations are made following the conventional
techniques of pharmacy involving milling, mixing, granulation, and
compressing, when necessary, for tablet forms; or milling, mixing and
filling for hard gelatin capsule forms. When a liquid carrier is used,
the preparation will be in the form of a syrup, elixir, emulsion or an
aqueous or non-aqueous suspension. Such a liquid formulation may be
administered directly p.o. or filled into a soft gelatin capsule.

[0080] The pharmaceutical compositions according to the invention may be
delivered in a therapeutically effective amount. The precise
therapeutically effective amount is that amount of the composition that
will yield the most effective results in terms of efficacy of treatment
in a given subject. This amount will vary depending upon a variety of
factors, including but not limited to the characteristics of the
therapeutic compound (including activity, pharmacokinetics,
pharmacodynamics, and bioavailability), the physiological condition of
the subject (including age, sex, disease type and stage, general physical
condition, responsiveness to a given dosage, and type of medication), the
nature of the pharmaceutically acceptable carrier or carriers in the
formulation, and the route of administration. One skilled in the clinical
and pharmacological arts will be able to determine a therapeutically
effective amount through routine experimentation, for instance, by
monitoring a subject's response to administration of a compound and
adjusting the dosage accordingly. For additional guidance, see Remington:
The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams
& Wilkins PA, USA) (2000).

[0081] Typical dosages of an effective quantity of pancreatic precursor
cells, insulin-producing cells derived from whole bone marrow, BMSC
expressing VEGF and/or PDX-1, or insulin-producing β-cells derived
from BMSCs can be as indicated to the skilled artisan by the in vitro
responses or responses in animal models. Such dosages typically can be
reduced by up to about one order of magnitude in concentration or amount
without losing the relevant biological activity. Thus, the actual dosage
will depend upon the judgment of the physician, the condition of the
patient, and the effectiveness of the therapeutic method based, for
example, on the in vitro responsiveness of the relevant primary cultured
cells or histocultured tissue sample, such as biopsied malignant tumors,
or the responses observed in the appropriate animal models, as previously
described.

[0082] The present invention is also directed to kits to produce
pancreatic precursor cells or insulin-producing cells derived from whole
bone marrow, and kits to treat diabetes. The kits are useful for
practicing the inventive method of producing pancreatic precursor cells
or insulin-producing cells derived from whole bone marrow and treating
diabetes. The kit is an assemblage of materials or components, including
at least one of the inventive compositions. Thus, in some embodiments the
kit contains a composition including pancreatic precursor cells or
insulin-producing cells derived from whole bone marrow, as described
above.

[0083] The exact nature of the components configured in the inventive kit
depends on its intended purpose. For example, some embodiments are
configured for the purpose of treating diabetes; some embodiments are
configured for the purpose of producing pancreatic precursor cells or
insulin-producing cells derived from whole bone marrow. In one
embodiment, the kit is configured particularly for the purpose of
treating mammalian subjects. In another embodiment, the kit is configured
particularly for the purpose of treating human subjects. In further
embodiments, the kit is configured for veterinary applications, treating
subjects such as, but not limited to, farm animals, domestic animals, and
laboratory animals.

[0084] Instructions for use may be included in the kit. "Instructions for
use" typically include a tangible expression describing the technique to
be employed in using the components of the kit to effect a desired
outcome, such as to produce pancreatic precursor cells or
insulin-producing cells derived from whole bone marrow, or to treat
diabetes. Optionally, the kit also contains other useful components, such
as, diluents, buffers, pharmaceutically acceptable carriers, syringes,
catheters, applicators, pipetting or measuring tools, bandaging materials
or other useful paraphernalia as will be readily recognized by those of
skill in the art.

[0085] The materials or components assembled in the kit can be provided to
the practitioner stored in any convenient and suitable ways that preserve
their operability and utility. For example the components can be in
dissolved, dehydrated, or lyophilized form; they can be provided at room,
refrigerated or frozen temperatures. The components are typically
contained in suitable packaging material(s). As employed herein, the
phrase "packaging material" refers to one or more physical structures
used to house the contents of the kit, such as inventive compositions and
the like. The packaging material is constructed by well-known methods,
preferably to provide a sterile, contaminant-free environment. The
packaging materials employed in the kit are those customarily utilized in
cell therapy. As used herein, the term "package" refers to a suitable
solid matrix or material such as glass, plastic, paper, foil, and the
like, capable of holding the individual kit components. Thus, for
example, a package can be a glass vial used to contain suitable
quantities of an inventive composition containing, whole bone marrow,
bone marrow derived neuropheres, pancreatic precursor cells derived from
whole bone marrow, insulin-producing cells derived from whole bone
marrow, BMSC expressing VEGF and/or PDX-1, or insulin-producing
β-cells derived from BMSCs. The packaging material generally has an
external label which indicates the contents and/or purpose of the kit
and/or its components.

EXAMPLES

[0086] The following examples are provided to better illustrate the
claimed invention and are not to be interpreted as limiting the scope of
the invention. To the extent that specific materials are mentioned, it is
merely for purposes of illustration and is not intended to limit the
invention. One skilled in the art may develop equivalent means or
reactants without the exercise of inventive capacity and without
departing from the scope of the invention.

Example 1

Cell Isolation and Culture

[0087] Adult Fisher rats were purchased from Charles River Laboratories
(Wilmington, Mass., USA). They were killed by CO2 asphyxiation
according to an approved protocol by the Institutional Animal Care and
Use Committee at Cedars Sinai Medical Center. Whole bone marrow was
harvested from the femurs as previously published (Talmor et al. 1998).
Briefly, the femurs were isolated from the muscle tissues and both ends
of the bones were cut. The marrow was flushed and the tissue was passed
through a mesh to remove small pieces of debris. After washing,
nestin-positive spheres were obtained as described previously by the
inventors' group (Kabos et al. 2002). Cells were plated at a density of
1×106 cells/well in poly-D lysinecoated 24-well plates (BD
Biosciences, San Jose, Calif., USA) in serum-free DMEM/F12 medium
(Invitrogen), supplemented with 20% B27 (Invitrogen), 20 ng/ml of
fibroblast growth factor (bFGF, Peprotech, Rocky Hill, N.J., USA), 20
ng/ml of epidermal growth factor (EGF, Peprotech), penicillin (100 U/ml)
and streptomycin (100 μg/ml). Medium was changed every 2 days. Cells
were maintained at 37° C. in a humidified atmosphere containing 5%
CO2. Portions of cells from sphere forming were dissociated to
single cells, frozen down (10% dimethylsulfoxide and 40% fetal bovine
serum) by cryotank in -80° C. for overnight and put into the
liquid nitrogen (LN2) tank for maintenance of cell populations.

[0088] For control experiment rat bone marrow mesenchymal stem cells
(MSCs-BM) were cultured on a-MEN medium (Invitrogen) supplemented with
10% FCS (Invitrogen). Briefly, the cells collected from bone marrow (as
described earlier) were plated into tissue culture-treated dishes of 100
mm in diameter at 37° C. in a humidified atmosphere containing 5%
CO2. After 24 h, floating cells were washed out and only adherent
cells were maintained in the culture with medium changed every 3 days.
Cells which reached 80-90% confluency in 100 mm dish were passaged to 1:4
and cultured in the same medium.

Example 2

Cell Culture and Differentiation

[0089] The inventors' group previously described the successful selection,
from rat whole bone marrow, of multipotent stem cells expressing nestin
(n-BMSC) and forming a spheroid structure with phenotypic characteristics
and a genetic signature typical of neuropheres (Kabos et al. 2002).

[0090] The inventors established a complex differentiation protocol
comprising four basic steps (FIG. 1). Control experiments were conducted
in parallel using rat MSCs-BM. In the first step (chromatin remodeling),
nestin-positive spheres were plated at a concentration of about 100
spheres/ml in each well of 6-well culture plate (Corning, Corning, N.Y.,
USA), in a basal medium containing KO-DMEM and main components: 0.1 mM
β-mercaptoethanol, 2 nM L-glutamine, 1% nonessential amino acid,
0.2% BSA, N2 supplement, B27 supplement (all from Invitrogen), 2 μg/ml
heparin, 20 ng/ml bFGF, and 20 ng/ml EGF. The cells were treated with 1
μM 5-AZA (Sigma) for 24 h. After 24 h the medium was changed and cells
were treated with 100 nM TSA (Sigma) for 24 h.

[0091] In the second step (induction), the basal medium was changed with
induction medium containing DMEM with low glucose (1 g/l; Invitrogen),
1× insulin-transferin-selenium (ITS; Invitrogen), 2 μM all-trans
RA (Sigma), and main components. The cells were maintained in induction
medium for about 7 days. In the third step (differentiation), the cells
were seeded into 6-well plates coated with poly-L-ornithine (15 μg/ml;
Sigma) at a concentration of 2-5×105 cells/well in
differentiation medium containing DMEM with high glucose (Invitrogen) and
main components for 7 days. In the last step (maturation) the medium was
modified from differentiation medium, adding 10 mM nicotinamide (Sigma)
without supplement of bFGF and EGF for 7 days. For each step the medium
was changed every 2 days.

Example 3

Quantitative Real-Time PCR

[0092] Cells were harvested at different steps and total RNA was extracted
using RNeasy Minikit (Qiagen), according to the manufacturer's
instruction. cDNA was prepared using Superscript reverse transcriptase
(Invitrogen). cDNA samples derived from 50 ng of total RNA was analyzed
by quantitative reverse transcription-PCR (qRT-PCR) using SYBR green dye
with QuantiTect SYBR Green RT-PCR kit (Qiagen).

[0093] Sequences of PCR primers used are listed in Table 1. PCR was
performed with cycles at 95° C. for 15 s, 56-60° C. for 10
s (Table 1) and 72° C. for 20 s. Reactions (40 cycles) were
carried out with iCycler PCR machine (Bio-Rad) and data analysis was
performed with QPCR software (Applied Biosystems, Foster City, Calif.,
USA). Each experiment was performed at least three times. Relative
quantitative analysis was performed following
2.sup.-ΔΔCT. The expression of each gene was normalized
to b-actin gene expression.

[0094] Spheroids were fixed in 4% paraformaldehyde in phosphate buffer for
10 min. Blocking was carried out for 30 min using 10% FCS diluted in PBS
at room temperature. The cells were then incubated with primary
antibodies in solution of 3% of FCS in PBS for an hour at room
temperature. Antibody dilutions were as follows: mouse monoclonal
anti-nestin (MAB353) (1:100; Millipore Corporate, Bellerica, Mass., USA),
goat anti-PDX1, rabbit anti-PAX6, rabbit anti-CK19, rabbit anti-insulin,
goat anti-glucagon, and rabbit anti-amylase (All 1:50, from Santa Cruz
Biotechnology, Santa Cruz, Calif., USA). Afterwards, the cells were
washed with PBS for 5 min for three times. Secondary antibodies were FITC
or Texas red conjugated anti-mouse, anti-goat and anti-rabbit (Vector
Laboratories, Inc., Burlingame, Calif., USA) and were diluted 1:500 in
PBS containing 3% of FCS. Cells were incubated in this solution for 45
min at room temperature, followed by washes in PBS for 5 min for three
times. Matched exposure of control sample was stained using non-immune
calf serum in place of the primary antibodies. Nuclear DNA was stained
with 4',6-diamidino-2-phenylindole (Vector Lab). Images were captured by
digital camera connected with fluorescent microscope (Model Upright
Zeiss, Jena, Germany). Scanning confocal images were obtained by a
confocal microscope (Leica Microsystems SP5, Mannheim, Germany).

Example 5

Dithizone Staining

[0095] Dithizone (DTZ, Sigma), which stains zinc-containing cells bright
red, was used to quickly assess the presence of insulin-producing cells.
The staining protocol was followed from the study by Shiroi et al.
(2002). DTZ stock solution was prepared by solving 50 mg of DTZ in 5 ml
of dimethyl sulfoxide (Sigma), sterile-filtering through a 0.22 μm
nylon filter, and stored at -20° C. The working solution was
prepared (pH 7.8) by diluting the stock solution 1:100 in culture medium.
For each dish, 2 ml of DTZ solution was added and incubated for 30 min at
37° C. After washing the cells three times in PBS, the
differentiated islet-like clusters were examined under phase contrast
inverse microscope.

Example 6

Insulin Assay

[0096] For each determination, about 100 spheroids of similar size were
randomly handpicked at stage 4 (3 weeks after starting the pancreatic
differentiation) and incubated in DMEM with low glucose (1 g/l) without
serum overnight. For insulin secretion assays spheroids were preincubated
for 1 h in Krebs-Ringer/bicarbonate buffer (KRB: 120 mM NaCl, 5 mM KCl,
2.5 mM CaCl2, 1.1 mM MgCl2, and 25 mM NaHCO3) at
37° C. or on ice (for control experiments) in 5% CO2
atmosphere, followed by incubation for an additional hour in KRB
containing 0.5 mM 1-isobutyl-3-methylxanthine and glucose at different
concentrations (5, 10, and 20 mM). The buffer was collected and frozen at
-70° C. until assay for insulin content. The amount of insulin
released was determined by ultrasensitive rat insulin ELISA (Linco
Research, Billerica, Mass., USA) according to the manufacturer's
protocol. Values showed the mean of three replicates ±S.D.

Example 7

Statistical Analysis

[0097] Results are shown as mean±S.D. Student t-test was used to
compare between two groups. One-way ANOVA was used to compare among three
groups. In all statistical analyses, a P value of <0.05 was judged
statistically significant.

Example 8

[0098] To drive differentiation of bone marrow stem cells expressing
nestin towards pancreatic lineages, the inventors developed a culture
procedure comprising four steps (FIG. 9). In the first step the cells
were exposed to 5-AZA for 24 h and TSA for another 24 h. The dose of
5-AZA used in this study has been successfully used by Lefebvre et al.
(2009) to induce NGN3 expression and endocrine differentiation into the
PANC-1 human ductal cell line. The inventors determined the optimal
concentrations of TSA by testing cell survival, growth and death with
increasing doses from 10 nM to 1 mM. Concentrations of more than 100 nM
for TSA showed an increased cell death and reduced differentiation
capability (data not shown). The inventors rationalized that chromatin
remodeling is the first step to induce nestin-positive cells because of
putative cell lineage differences and specification, and believed that
the increased `susceptibility` of chromatin by serial epigenetic
modifications will enhance the induction and further direct
differentiation toward the pancreatic phenotype (FIG. 9). After exposure
to TSA, the cells were cultured for 7 days in a serum-free medium
containing ITS and RA (FIG. 9C, induction). The concentration of RA
adopted in this study was previously optimized in embryonic stem cell
differentiation model (Cai et al. 2009). Recently, the role of RA in
pancreatic development and early endocrine lineage determination has been
shown (Ostrom et al. 2008). In addition, RA has been used for both mouse
and human embryonic stem cells to induce differentiation into pancreatic
cell lineage (Micallef et al. 2005, Johannesson et al. 2009). At this
time point the inventors were able to highly induce the expression of a
pancreatic master gene (pancreas duodenum homeobox-1, PDX1), together
with other genes important in the early pancreatic development (PAX6 and
NGN3). Many cells were positive for PDX1 as assayed by
immunohistochemistry (FIG. 10F). Subsequently, the PDX1-positive cell
population dramatically decreased after 7 days during the third step
(differentiation; FIG. 10G) but increased again and became prominent in
the fourth step (maturation; FIG. 10H). This fluctuation of PDX1-positive
cell population by immunohistochemistry also appeared in the qRT-PCR
analysis in sequential manner (FIG. 11B). Pdx1 gene expression was
initiated within 7 days during the second step culture (induction), then
downregulated several fold but was detectable ˜2 weeks later during
the third step, and upregulated again 3 weeks later during the maturation
step. Ngn3 transcript was induced at day 7 and progressively increased
two- to fivefold at day 14 (step 3) and day 21 (step 4) respectively
(FIG. 11). Only a few cells were positive for PAX6 at day 9, as confirmed
by immunohistochemistry (FIG. 10) and the number of positive cells
strongly increased during step 3, but was not detectable at step 4.
Surprisingly, these results were consistent with the physiological
expression of PDX1, PAX6, and NGN3 genes during in vivo pancreas
development. In contrast, nestin expression was high in the beginning of
the differentiation and dramatically decreased to an undetectable level
during the second stage of conditioning. This was confirmed by both
immunohistochemistry and (FIG. 10A-D) and qRT-PCR (Table 2).
Morphologically, the cells stayed aggregated in spheroid structure.

[0099] In the third stage of conditioning the inventors used
poly-Lornithine-coated dishes with a medium containing high glucose with
both bFGF and EGF. In such an environment, the cells underwent further
differentiation into a pancreatic phenotype. At about 2 weeks of the
differentiation process, corresponding to the maximized expression of
PAX6, increased expression of NGN3 and downregulation of PDX1, endocrine
markers for a more mature phenotype started to appear as confirmed by
qRT-PCR: insulin, glucagon, and glucose transporter 2 (Glut2; Table 2).
The expression of Ck19, a ductal marker, was also induced. At this stage
immunohistochemistry was negative for the same markers (FIG. 12).

[0100] In the last step, the inventors tried to further push the
differentiation toward a more mature phenotype. For this reason the
inventors used nicotinamide for about 7 days. At day 21 from the
beginning of the in vitro differentiation, a mixed population of cells
expressing endocrine and ductal phenotypes were observed.
Immunofluorescent staining was markedly positive for insulin and CK19
(FIG. 12). These cells also positively stained with the zinc chelator,
DTZ, indicating the presence of intracellular proinsulin, as has been
previously observed for `pseudoislets` in culture (Kuo et al. 1992). Only
a small number of cells were positive for glucagon with immunofluorescent
staining (data not shown). Insulin, CK19, and glucagon transcripts were
also consistently upregulated (seven- to eightfold, five- to sixfold and
two- to threefold, respectively; FIGS. 13A and 13B). No amylase was
detected on immunohistochemistry (data not shown) and RNA levels (Table
2), indicating the absence of a pancreatic exocrine phenotype. In this
culture condition the inventors also failed to detect somatostatin and
pancreatic polypeptide-positive cells (Table 2).

[0102] To evaluate the functionality of differentiated cells, the
inventors tested for glucose-induced insulin secretion in vitro. At the
end of the last differentiation stage, the cell aggregate (spheroids) not
only produced insulin, but also displayed a glucoseresponsive secretion
of the hormone. Insulin secretion was dose-dependent after incubation for
1 h with glucose (5, 10, and 20 mM; FIG. 13C). Spheroids incubated on ice
under the same condition did not show glucose-responsive insulin
secretion. The average insulin secretion on ice with glucose 5, 10, and
20 mM was 0.85±0.32, 1.5±0.41, and 1.8±0.5 ng/100 spheroids per
hour respectively. This is consistent with inhibition of glucose-induced
insulin release by cooling (Atwater et al. 1984). These findings
demonstrate de novo synthesis and processing of insulin and
physiologically regulated secretion. Moreover the cells expressed a
functional element, Glut2 (FIGS. 13A and 13B) that allows the rapid entry
of glucose into the cells. This confirmed the capability of the
inventors' differentiated cells to respond to glucose stimulation.

Example 9

Human BMSC Culture and Expansion

[0103] Bone marrow aspirate from normal adult donor was purchased from
Allcell (Emeryville, Calif.). All of the nucleated cells were plated in
100-cm2 culture dish (Corning, Corning, N.Y.) in a basal medium
consisting of Alfa-Men, 17% fetal bovine serum, 2 mM glutamine, 50 U/L
penicillin and 25 μg/L streptomycin (all from Invitrogen). After 24 hr
in culture, non-adherent cells were separated from adherent cells. The
adherent cells were washed with PBS and cultured with the previous medium
for 5-7 days. At 80% confluences the cells were harvested with
trypsin/EDTA and plated in two 100-cm2 dishes (passage 1). Cell
underwent two further expansions before aliquots were storage in 90% FBS
and 10% DMSO and frozen in -80° C. Until passage 6-7 the cells
were growing with doubling time of 48 hrs. All the experiments were
performed using a single batch of hBMSCs from a single donor. The cells
were used within passage 7. Human BMSCs from passage #3 to 6 were
analyzed for various cell surface markers commonly used for the positive
and negative detection of mesenchymal stem cells by flow cytometry
analysis (CD44, CD31, CD34, and CD 105). To characterize the mesenchymal
potential of hBMSCs, the inventors induced adipogenic and osteogenic
differentiations in vitro (data not shown).

Example 10

Flow Cytometric Analysis

[0104] Human BMSCs were analyzed for surface expression of different
markers commonly used for the positive and negative detection of
mesenchymal stem cells from passage 3 to 6. Cells were detached with
tripsin/EDTA and incubated with respective fluorochrome-conjugated
antibodies for cell surface glycoproteins: CD44, CD31, CD34, and CD 105
(BD Biosciences, San Diego). Fluorochrome-conjugated Isotype IgG were
used as control. Cells were immediately analyzed by BD FACScan and
analyses were done using BD CellQuest software (BD Biosciences), and
percent statistics were given.

Example 11

Adenovirus Production and Cell Infection

[0105] c-DNAs encoding for human Pdx1 and mouse VEGF165 were subcloned
into Adeno-X viral DNA vector (BD Biosciences Clontech), following
manufacturing protocol #PT3414-1, Version #PR31147. CMV has been used as
promoter. Successful homologous recombination resulted in recombinant
virus encoding for PDX1 (Ad-PDX1) and VEGF (Ad-VEGF). The virus was
expanded in HEK293 cells as described in the ViraPower Adenoviral
Expression system manual by Invitrogen. The viral titer was determinate
using a kit from Clontech and found a pfu of ˜1.1×10 E 9 for
all 2 constructs. Human BMSCs were transfected with adenovirus carrying
PDX1 (hMSC-BM-Pdx1) or carrying VEGF (hMSC-BM-VEGF) 2 days before
transplantation. RNA and protein level of PDX1 and VEGF in the
transfected cells were assessed by PCR and Western Blotting.

Example 12

In Vivo Animal Model and Stem Cell Transplantation

[0106] To induce diabetes, NOD/scid mice (The Jackson Laboratory, Bar
Harbor, Me.) 6-8 weeks of age were given three intraperitoneal injections
of streptozotocin (STZ) [2-Deoxy-2-(3-methyl
1-3-nitrosoureido)-D-glucopyranose, STZ; Sigma-Aldrich, Saint Louis,
Mo.], 50 mg/kg, on day 1-3. All experiments and procedures were performed
according to an approved protocol by the Institutional Animal Care and
Use Committee at Cedars Sinai Medical Center. STZ was dissolved in sodium
citrate buffer, pH 4.5, and injected within 15 minutes into fasted mice.
A total of 36 mice were used for this study. One control group (n=4 mice)
did not received any treatment, another group (n=6) received a sham
injection after induction of diabetes with STZ. The other 3 groups
received stem cell transplantation after induction of diabetes, hBMSC
(n=6), h-BMSC-Pdx1 (n=8), or h-BMSC-Vegf (n=9) respectively.
Additionally, two groups of STZ treated mice were transplanted with mouse
fibroblasts transfected with adenovirus expressing PDX1 or VEGF.

[0107] On day 0, about 7 days from STZ treatment, mice were transplanted
with about 1×106 cells each. To avoid aggregation of the
cells, hBMSC were suspended in 150 μl and injected though the chest
wall into the left cardiac ventricle as previously described [7], using a
30 gauge needle. Cells were infected with adenovirus vector 2 days prior
to injection into recipient mice. Mouse skin fibroblasts were used as
control and transfected with adenovirus expressing PDX1 and VEGF and
transplanted in similar conditions (4 mice for each group). All the
animals were sacrificed at 6 weeks after stem cell transplantation and
peripheral blood, and tissues were collected. Achievement of
normoglycemia was defined as blood glucose <200 mg/d1.

Example 13

Blood Glucose and Serum Insulin Measurements

[0108] Blood glucose was measured in non-fasten mice between 9 and 11 am
two times a week. The level of glucose was measured from the tail vein
using One Touch Ultra Meter and Test Strips (Lifescan Inc., Milpitas,
Calif.). The sensitivity of the assay does not exceed 600 mg/dl, so the
maximal extent of hyperglycemia maybe greater than indicated. Mouse serum
insulin was determinate by ultrasensitive mouse insulin enzyme-linked
immunoabsorbent assay (ELISA) (Alpco Diagnostics, Salem, N.H.) and human
serum insulin level by human insulin ELISA (Linco Research, Millipore
Corporation, Billerica, Mass.) according to the manufacturer's protocol
at 6 weeks after stem cell injection in fasten animals. Three replicates
for each sample were used.

[0110] Briefly, the pancreatic sections were sequentially incubated with
anti-insulin/proinsulin mouse monoclonal antibody (Abcam; dilution
1:200), biotinylated rabbit anti-guinea pig antibody (1:200 dilution;
Santa Cruz), and streptavidin-alkaline phosphatase complex (Santa Cruz),
for a period of 45 min each. The alkaline phosphatase activity was
identified using new fuchsin under light microscope. The sections were
counterstained with Harris hematoxylin. The images of the above stained
sections were captured at 100× magnifications. The inventors
selected 3 sections separated from 200 μm and counted the number of
insulin expressing cells in all 3 sections. The inventors used
immunofluorescent staining and confocal imaging to quantify β-cells
expressing VEGF, human β2-microglobulin, and caspase 3 cleaved. The
inventors used pancreas from 3 mice from each group.

[0112] Pancreatic tissues were preserved with RNA Later (Invitrogen) at
-20° C. Total RNA was extracted using RNeasy Minikit (Quiagen),
according to the manufacturer's instruction. mRNA from healthy control
mice, STZ diabetic mice and STZ diabetic mice rescued by hBMSC-VEGF were
obtained as previously described for the Real Time PCR. Briefly, RT2
First Strand Kit (SABiosciences, Frederick, USA) was used to convert mRNA
to cDNA. This cDNA was then added to the SAbiosciences RT2 SYBR Green
qPCR Master Mix. Each sample was used to performed quantitative gene
expression analysis on specific arrays for the insulin signaling pathway
(Cat#PAMM-030-F). All steps were done according to the manufacturer's
protocol for the Roche Light Cycler 480. The online tool
(http://www.sabiosciences.com/pcrarraydataanalysis.php (last accessed
Aug. 1, 2011) offered by the manufacturer was used to analyze the data,
including significant value and fold changes. Each set of data was
repeated 4 times. Only significant results (p<0.05) were taken into
consideration.

Example 17B

DNA Extraction and Real-Time PCR

[0113] Frozen tissues were homogenized, and genomic DNA was extracted
using DNeasy Blood & Tissue Kit (Qiagen) from mouse organs and human
BMSCs. Mouse DNA was isolated from identical tissues of non-transplanted
NOD/SCID mice as used as negative control. In addition, human DNA was
isolated from hBMSC cultures and used as positive control. Total DNA was
assayed by UV absorbance. Real time-PCR was performed with 100 ng target
DNA. To detect human DNA in the mouse tissues the inventors used
previously reported human specific primers, targeting a unique and
conserved region of human β-actin [26]. Endogenous mouse GAPDH gene
(Qiagen, QT01658692) was also amplified as internal control. Real-Time
PCR was carried out with iCycler (Bio-Rad), using QuantiFast SYBR Green
PCR kit (Qiagen), following manufacture protocol. Absolute standard
curves were obtained for the human β-actin and mouse GAPDH. To
evaluate human specificity of human β-actin gene, standard curves
were generated by serially diluting human genomic DNA in mouse DNA.
Values are expressed in percent of human DNA infused as cells in the
mouse tissue. Each assay was carried on in triplicates and repeated at
least 3 times.

Example 18

Statistical Analysis

[0114] All data were presented as mean±SD and were compared by student
T-test. P<0.05 was considered to indicate statistical significance of
the test results. Kaplan-Meier curves were used for the survival study
and the log-rank (Maltel-Cox) test was used to determine statistical
significance. For multiple comparisons the inventors used one way ANOVA
followed by Tukey test.

[0117] Diabetes was induced in NOD/SCID mice with streptozotocin (STZ), a
cytotoxic agent that preferentially damages β-cells. All mice
treated with STZ (n=6) developed hyperglycemia 6-7 days after STZ
injection and 50% of them died before 6 weeks (FIG. 1A). Control
untreated mice (n=5) maintained euglycemia during the study period (FIG.
1A). The inventors tested the ability of hBMSCs genetically modified to
transiently express VEGF gene to rescue diabetic mice. These cells were
injected into the circulation of the STZ-induced diabetic mice (n=9) one
week after STZ treatment. Reversion of hyperglycemia was observed in 5 of
9 mice treated with hBMSC-VEGF between 1 to 2 weeks after cell injection
and near-normoglycemia remission was maintained for 6 weeks (FIG. 1B,
`rescued`). Four of the mice treated with hBMSC-VEGF failed to reverse
hyperglycemia (`unrescued`).

[0118] Although divided in two groups, rescued and unrescued, all mice
treated with hBMSC-VEGF showed better clinical outcomes in terms of
survival rates and weight gain compared with the diabetic STZ treated
mice (FIG. 1C,D). All of rescued and unrescued groups of mice gained
weight significantly, which is comparable to the healthy control mice
(FIG. 1C). These mice survived by the end time of the study in contrast
to the high mortality rate of the diabetic mice (FIG. 1D, p<0.05).
Overall, the response to treatment in terms of reversion of hyperglycemia
was significantly higher in the mice treated with hBMSCs-VEGF compared
with the mice receiving sham injection (p=0.025).

[0119] Histological examination at 6 weeks from transplantation showed
severe alteration of the pancreatic islet morphology and significant
reduction of the number of insulin-expressing cells in the STZ-induced
diabetic mice (FIG. 1F,J) and in the unrescued mice (FIG. 1H,L). In
contrast, the morphology of pancreatic islet was maintained and the
staining patterns of insulin in the pancreatic islets of hBMSCs-VEGF
treated mice (FIG. 1G,K) were very similar to one in the healthy control
mice (FIG. 1E,I). Further investigation showed that hBMSCs-VEGF were
robustly engrafted and diffusely survived in the pancreas (FIG. 2A-B),
while fewer human cells were present in the pancreas of the unrescued
animals at 6 weeks after transplantation (FIG. 2C). Engraftment and
survival of hBMSCs-VEGF in the mouse pancreas was also assessed by
real-time PCR for a human-specific gene (Table 1). The pancreatic samples
from mice treated with hBMSC-VEGF showed variable amount of human DNA.
Small amount of DNA was also present in the kidney of few mice but not in
other organs (Table 1).

[0120] Furthermore hBMSCs-VEGF were able to differentiate into vessels and
β-cells as confirmed by co-staining with human β2-microglobulin
which specifically stained human cells and either a-smooth muscle actin
or insulin (FIG. 2D). Vascular differentiation was prominent inside the
pancreas, however, the efficiency of differentiation into β-cells
was low. It is interesting to note that only a small percentage of
pancreatic islets were positive for human β2-microglobulin (FIG.
2E). However, those pancreatic islets containing human cells showed
approximately half of the β-cells originated from human (FIG. 2E,
right panel).

[0121] In the healthy age matched control mice, VEGF was uniquely
expressed only in the pancreatic isles, mostly by the β-cells (FIG.
3A-C). After induction of diabetes with STZ, the inventors observed a
dramatic and significant reduction of VEGF expression in the β-cells
(FIG. 3D-F), which was completely restored after treatment with
hBMSC-VEGF (FIG. 3A-J).

[0122] Further investigation showed that hBMSC-VEGF robustly engrafted and
diffusely survived in the mouse pancreas and the mesenchymal tissue
surrounding the pancreas of the rescued mice (FIG. 2F-G), while fewer
human cells were present in the pancreas of the unrescued animals at 6
weeks after transplantation.

[0123] The inventors tried to determine if hBMSCs genetically modified to
express PDX1 (hBMSCs-PDX1) were able to rescue diabetic mice. The cells
were injected into the circulation of the diabetic mice (n=8) 7 day after
STZ treatment. Four mice showed reduction of the hyperglycemia in the
following week after transplantation (`temporary reversed`), while 4 mice
maintained severe hyperglycemia (`unrescued`, FIG. 4A). Interestingly,
the rescued mice maintained near-normoglycemic remission for 2-3 weeks,
and then developed severe hyperglycemia again. All mice (`temporary
reversed` and `unrescued`) survived at 6 weeks from transplantation,
compared with the significant drop in survival rate of the diabetic mice
(FIG. 4B, p<0.05), and gained significant weight compared with the
diabetic control mice (FIG. 4C), indicating a better clinical outcome.
Isolated pancreas from `temporary reversed` as well as `unrescued` mice
analyzed for immunostaining against insulin showed reduction of insulin
expression in the pancreatic islets compared (FIG. 4F,G) with healthy
control mice (FIG. 4D) and similar to the STZ-treated diabetic mice (FIG.
4E). Staining for human beta-2 microglobulin clearly showed the
engraftment of human cells in pancreases (FIG. 4H). In both groups
(`temporary reversed` and `unrescued`), the inventors found none of
vessel-like structures were differentiated from the hBMSCs-PDX1. A few
transplanted cells expressing insulin in islet structures were noted as
shown in FIG. 4H, implicating functional differentiation into
β-cells.

[0124] In contrast with the two above results, hBMSCs that were not
genetically modified were not able to ameliorate the diabetes phenotype.
Diabetic mice (n=6) treated with one intra-left ventricular injection of
1×106 hBMSCs at day 7 from STZ injection continued to maintain
severe hyperglycemia (FIG. 5A). Control experiments were carried on with
fibroblasts expressing VEGF or PDX1 and did not show improvement of
hyperglycemia after fibroblast transplantation (FIG. 5B). More than 50%
of the mice treated with hBMSCs died before 6 weeks showing a survival
rate similar to the STZ-induced diabetic mice and significantly lower
than the healthy control mice (FIG. 5C, p<0.05). In addition survived
mice that survived failed to gain weight (FIG. 5D) at 6 weeks
post-transplantation. Histological examination showed severe alteration
of the pancreatic islet morphology in this group (FIG. 5E) similar to the
STZ-induced diabetic mice. The pancreatic islets of mice receiving hBMSC
showed reduction in insulin expression with the characteristic inversion
in the ratio insulin/glucagon cells (FIG. 5F). Further investigation
showed poor engraftment of hBMSCs in the pancreas at 6 weeks after
transplantation (FIG. 5G). Detection of human DNA in the mouse pancreas
at 6 weeks post-transplantation was low (Table 3). The inventors were
able to detect human DNA in two out of four tested mice with a lower
concentration compared with the mice treated with hBMSC-VEGF, confirming
a lower engraftment and/or survival of donor cell in the recipient
pancreas.

[0125] To evaluate the possible contribution of hBMSC-VEGF to the recovery
of β-cells and whether the reversion of diabetes was secondary to a
direct differentiation of hBMSC to β-cells or secondary to
endogenous β-cell regeneration, the inventors measured both mouse
and human serum insulin. Only the mice rescued by hBMSC-VEGF had
significantly higher level of mouse insulin compared with that of
diabetic mice and diabetic mice treated with hBMSC or hBMSC-PDX1 (FIG.
6A). However, more than half of the mice treated with hBMSC-VEGF and
hBMSC-PDX1 (5 out of 9 and 5 out of 8, respectively), showed low but
detectable level of human insulin, confirming de novo human BMSC
differentiation into functional β-cells (FIG. 6B). The levels of
total serum insulin were significantly higher in the mice treated with
hBMSC-VEGF and hBMSC-PDX1 compared with the diabetic mice and the
diabetic mice treated with hBMSC (FIG. 6C). The result showed clear
correlation between the level of insulin and the number of β-cells,
which was again higher in the mice treated with hBMSC-VEGF and hBMSC-PDX1
than two other groups (FIG. 6D). In addition, even unrescued groups of
mice from hBMSC-VEGF and hBMSC-PDX1 with persistent hyperglycemia
demonstrated significantly higher levels of total serum insulin and
numbers of endogenous β-cells compared with other groups resulting
in the overall better clinical outcomes. However, the group of mice with
sustained near-normoglycemia remission, treated with hBMSC-VEGF, had the
highest level of mouse insulin and β-cell numbers among the groups,
suggesting that sustained reversion of diabetes was secondary to
endogenous β-cell regeneration or recovery rather than
transplant-derived β-cell differentiation.

Example 21

Endogenous β-Cell Recovery in Mouse Rescued by hBMSC-VEGF was
Secondary to Activation of Insulin/IGF Signaling Pathway

[0126] To evaluate the possible mechanism of endogenous β-cell
recovery mediated by hBMSC-VEGF in the rescued mice the inventors
compared the PCR array data of the pancreases from the healthy and
diabetic age matched control mice. The inventors noted a clear trend of
decreasing expression of mouse genes related with insulin receptor
signaling pathway in pancreases of the diabetic mice compared with the
healthy control (FIG. 7A). In the STZ-induced diabetic mice group, there
was a significant decrease in expression of Insulin1, as expected. In
addition, a significant decrease in gene expression of Excision repair
cross-complementing rodent repair deficiency 1 (Ercc1), Glucokinase
(Gck), and Acetyl-Coenzyme A carboxylase alpha (ACACA) with an increased
expression of Jun. However, mice rescued by hBMSC-VEGF showed a similar
PCR-array profile as seen in the healthy control mice without significant
changes in gene expression (FIG. 7B). Interestingly, PCR array of the
diabetic mice rescued by hBMSC-VEGF showed the significant up-regulation
of the mouse genes involved in the insulin/IGF signaling pathway as
compared to the diabetic mice (FIG. 7C). In particular, insulin was
significantly upregulated in the rescued mice while it was downregulated
in the STZ-induced diabetes mice as compared to the healthy control.
Insulin receptor associate protein such as insulin growth factor 2
(IGF2), insulin growth factor binding protein 1 (Igfbp1), and Dok3 were
significantly upregulated in the rescued mice. All target genes for
phosphatidylinositol 3-kinase (PI-3K) pathway such as Adrenergic receptor
alpha1d (Adra1d), glucose-6-phosphatase catalytic (G6 pc),
glucose-6-phosphatase, catalytic, 2(G6pc2), and Serpine 1 were also
upregulated while Eukaryotic translational initiation factor 2B, subunit
1 (Eif4ebp1), Growth factor receptor bound protein 2 (Grb2) and Jun were
significantly downregulated as compared to the diabetic mice (FIG. 7C).

[0127] To further explore the molecular mechanisms for the reversion of
diabetes and β-cell recovery/regeneration in diabetic mice treated
with hBMSC-VEGF, pancreatic islets from control healthy mice, STZ-induced
diabetic mice, and diabetic mice rescued by hBMSC-VEGF were examined
using high resolution confocal imaging to assess insulin/IGF
receptor/PI3-K downstream proteins. AKT protein was highly expressed in
the pancreatic islets of healthy mice mostly on the plasma membranes
(FIG. 8A, upper panel) while dramatically reduced in diabetic condition
(FIG. 8A, intermediate panel). Interestingly, after injection of
hBMSC-VEGF, pancreatic islets of rescued mice showed upregulation of AKT
expression with distribution not only in the plasma membrane but also
strongly in the cytoplasm (FIG. 8A, lower panel). Activation of
insulin/IGF receptor/PI-3K/AKT pathway is associated to increase
β-cell mass though activation of downstream proteins required for
β-cell proliferation, differentiation and survival, such as PDX1
[27,28] and P27Kip1 [29,30]. Associated with preservation and/or
regeneration of β-cell number and insulin secretion, mice rescued by
hBMSC-VEGF showed a strong nuclear localization of PDX1 (FIG. 8B lower
panel) similar to the control mice (FIG. 8B upper panel). In contrast
PDX1 was detected in less β-cell nuclei of the diabetic mice (FIG.
8B, intermediate panel), with a staining that appears to be weaker. In
addition, virtually all β-cell nuclei of the control mice were
positive for p27.sup.Kip1 (FIG. 8C, upper panel), a cell cycle inhibitor
protein negatively regulated through the PI-3K/AKT pathway. This is
consistent with the slow β-cell replication in postnatal life [31].
After induction of diabetes most β-cell nuclei were positive for
p27.sup.Kip1 (FIG. 8C, intermediate panel), suggesting a persistent
inhibition of β-cell replication. In contrast the inventors detected
a markedly decrease in immunostaining for p27.sup.Kip1 protein in the
pancreatic islets of mice rescued by hBMSC-VEGF, with only isolated
positive nuclei (FIG. 8C, lower panel), consistent with activation of
β-cell replication.

[0128] The inventors next measured caspase 3 cleaved (c-CASP3) to
β-cell assess apoptosis in relation to activation of the Insulin
receptor/PI-3K/AKT pathway. Pancreatic islets from diabetic mice showed a
dramatic increase in c-CASP3 compared with control and rescued mice (FIG.
8D), and the percent of c-CASP3-positive β-cells in diabetic mice
rescued by hBMSC-VEGF was significantly lower compared to the diabetic
mice and similar to the healthy control mice (FIG. 8E). This is
consistent with anti-apoptotic signal mediated by activation of PI-3K
pathway (FIG. 8F).

[0231] Various embodiments of the invention are described above in the
Detailed Description. While these descriptions directly describe the
above embodiments, it is understood that those skilled in the art may
conceive modifications and/or variations to the specific embodiments
shown and described herein. Any such modifications or variations that
fall within the purview of this description are intended to be included
therein as well. Unless specifically noted, it is the intention of the
inventors that the words and phrases in the specification and claims be
given the ordinary and accustomed meanings to those of ordinary skill in
the applicable art(s).

[0232] The foregoing description of various embodiments of the invention
known to the applicant at this time of filing the application has been
presented and is intended for the purposes of illustration and
description. The present description is not intended to be exhaustive nor
limit the invention to the precise form disclosed and many modifications
and variations are possible in the light of the above teachings. The
embodiments described serve to explain the principles of the invention
and its practical application and to enable others skilled in the art to
utilize the invention in various embodiments and with various
modifications as are suited to the particular use contemplated.
Therefore, it is intended that the invention not be limited to the
particular embodiments disclosed for carrying out the invention.

[0233] While particular embodiments of the present invention have been
shown and described, it will be obvious to those skilled in the art that,
based upon the teachings herein, changes and modifications may be made
without departing from this invention and its broader aspects and,
therefore, the appended claims are to encompass within their scope all
such changes and modifications as are within the true spirit and scope of
this invention. It will be understood by those within the art that, in
general, terms used herein are generally intended as "open" terms (e.g.,
the term "including" should be interpreted as "including but not limited
to," the term "having" should be interpreted as "having at least," the
term "includes" should be interpreted as "includes but is not limited
to," etc.).